Structural and biophysical analyses of the skeletal dihydropyridine receptor β subunit β_1a reveal critical roles of domain interactions for stability.

Excitation-contraction (EC) coupling in skeletal muscle requires a physical interaction between the voltage-gated calcium channel dihydropyridine receptor (DHPR) and the ryanodine receptor Ca²⁺ release channel. Although the exact molecular mechanism that initiates skeletal EC coupling is unresolved, it is clear that both the α₁ and β subunits of DHPR are essential for this process. Here, we employed a series of techniques, including size-exclusion chromatography-multi-angle light scattering, differential scanning fluorimetry, and isothermal calorimetry, to characterize various biophysical properties of the skeletal DHPR β subunit β_1a Removal of the intrinsically disordered N and C termini and the hook region of β_1a prevented oligomerization, allowing for its structural determination by X-ray crystallography. The structure had a topology similar to that of previously determined β isoforms, which consist of SH3 and guanylate kinase domains. However, transition melting temperatures derived from the differential scanning fluorimetry experiments indicated a significant difference in stability of ∼2-3 °C between the β_1a and β_2a constructs, and the addition of the DHPR α_1s I-II loop (α-interaction domain) peptide stabilized both β isoforms by ∼6-8 °C. Similar to other β isoforms, β_1a bound with nanomolar affinity to the α-interaction domain, but binding affinities were influenced by amino acid substitutions in the adjacent SH3 domain. These results suggest that intramolecular interactions between the SH3 and guanylate kinase domains play a role in the stability of β_1a while also providing a conduit for allosteric signaling events.

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